Int. J. Pharm. Sci. Rev. Res., 21(2), Jul – Aug 2013; nᵒ 11, 58-61
ISSN 0976 – 044X
Research Article
High Performance Liquid Chromatography Method for the Determination of
Terbinafine Hydrochloride in Semi Solids Dosage Form
Hamsa Kassem*, Mohamed Amer Almardini
Damascus University, Damascus, Syria.
Accepted on: 03-05-2013; Finalized on: 31-07-2013.
ABSTRACT
A reversed-phase high performance liquid chromatography (HPLC) method has been developed and validated to determine
terbinafine hydrochloride in semi solids dosage forms (creams). The chromatography was performed on C18 column and a mobile
phase consisting of methanol and acetonitrile (60:40 v/v) with (0,15% triethylamine and 0,15% phosphoric acid), eluents were
monitored by UV photo diode array detector at wave length of 224nm. The method was statistically validated for linearity,
specificity, accuracy, precision, limit of detection, limit of quantification and robustness.
Keywords: Liquid chromatography, Terbinafine hydrochloride, semi solid dosage form.
INTRODUCTION
T
erbinafine hydrochloride is a synthetic antimycotic
agent from a new class of compounds, the
allylamines (fig 1). It selectively inhibits fungal
squaline epoxidase causing a fungicidal action due to the
intracellular accumulation of the toxic sterol squaline, it
also exerts a fungistatic action by depletion of
ergosterol.1,2
pharmaceutical company, Damascus ‫ ـ‬Syria) which offered
also samples of terbinafine hydrochloride cream and all
solvents and reagents which were needed to the study.
THE creams were claimed to contain 1% of terbinafine
hydrochloride.
HPLC apparatus VWR Hitachi La Chrom Elite model with
auto sampler and diode array detector.
Standard solution preparation
Approximately (10) mg of terbinafine hydrochloride (RS)
accurately weighed was transferred to (100) ml
volumetric flask and diluted with methanol to the
volume. Then (10) mg of this solution was transferred to
(100) ml volumetric flask and diluted to the volume with
methanol. The final concentration of this solution was
considered to be (0, 01) mg of terbinafine hydrochloride.
Figure 1: Chemical structure of terbinafine hydrochloride.
Sample preparation
Terbinafine hydrochloride now is the drug of choice in
dermatophyte nail and skin infections because of its
fungicidal mode of action over a short treatment
3
duration.
A quantity of the cream containing (1) mg of terbinafine
hydrochloride was extracted with (25) methanol with the
help of sonicating, then this solution was transferred
quantitatively to (100) ml volumetric flask and methanol
was added to make up to volume. After sonicating for (5)
minutes a portion of the solution was filtered through a
filter having a porosity of 0, 45 nm. The final
concentration of terbinafine hydrochloride was
considered to be (0, 01) mg/ml.
There are few methods to determine terbinafine
hydrochloride in dosage forms and biological fluids, these
methods include microbiology4,5, electrochemistry6,
spectrophotometry7‫ـ‬11, titrimetry8, high performance thin
layer chromatography(HPTLC)12,9, high performance liquid
13‫ـ‬21
chromatography (HPLC)
and gas chromatography
22,23
(GC) , therefore the aim of our study was to develop a
simple and sensitive reversed ‫ـ‬phase (HPLC) method for
determination of terbinafine hydrochloride in semi solids
dosage forms (creams).
Methods
Chromatographic conditions
The chromatographic conditions which were used in the
method are displayed in table 1.
MATERIALS AND METHODS
Preparation of solutions for validation study
Materials
Solutions for linearity: Five sequential concentrations
were prepared containing 50%, 75%, 100%, 125% and
150%, respectively of the standard solution.
Reference terbinafine (assigned purity99, 9%) was kindly
supplied by al Phares Company (local private
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58
Int. J. Pharm. Sci. Rev. Res., 21(2), Jul – Aug 2013; nᵒ 11, 58-61
ISSN 0976 – 044X
Table 1: Chromatographic conditions of HPLC method.
Column
Mobile phase
Detection and
wave length
Flow rate
Injection volume
Oven temperature
Intersil : L1ODS(4,6mm.15cm)
particle size 5 µg
Methanol - acetonitrile (60:40
v/v) with (0,15% triethylamine
and 0,15% phosphoric acid )
PH=7,68
Photo diode array detector
monitored at 224 nm
0,4 ml/minute
10 µl
25°C
Solutions for selectivity: A drug ‫ـ‬free sample (placebo)
was prepared from the excipients. Six samples were
prepared also, three of them contain placebo and the
others do not contain placebo.
Solutions for accuracy: Cream excipients were individually
spiked with the standard solutions to obtain analysis
samples. Nine samples were divided into three groups
containing respectively 50%, 100%and 150% of the
corresponding standard solution.
Solutions for precision: Nine samples of terbinafine
hydrochloride cream covering the working range 50%
to150% were prepared.
Figure 2: HPLC chromatogram for terbinafine
hydrochloride by using C18 column and a mobile phase
consisting of methanol – acetonitrile (60:40 v/v) with
(0,15% triethylamine and 0,15% phosphoric acid)
PH=7,68.
Range and linearity
A series of terbinafine hydrochloride concentrations
containing 50%‫ـ‬150% of the standard concentration (0, 01
mg/ml) was injected in HPLC apparatus and the analyses
were performed triplicate. By drawing the regression line
for peak area versus the concentration linearity of the
method was proved (fig 3).
The equation of the regression line was: y= 1E + 0,9x 594568 and the correlation coefficient was 0, 9993.
Solutions for robustness: Samples of terbinafine
hydrochloride cream containing 100% of standard
solution were prepared.
RESULTS AND DISCUSSION
Chromatographic conditions
It was noticed that lipophilic compounds with alkalyted
nitrogen can easily be separated on reversed ‫ـ‬phase
column using acetonitrile as a mobile phase and if
triethylamine is added, peak tailing due to analyte
interaction with free silanol groups could be avoided. In
our study a mobile phase consisting of water –
acetonitrile (60:40 v/v) with (0,15% triethylamine and
0,15% phosphoric acid) was tested first and the
chromatography was performed on C18 column and the
eluents were monitored with photo‫ ـ‬diode array UV
detector at 224 nm wave length. By using these
chromatographic conditions a peak of terbinafine
hydrochloride appeared at a retention time of 4 minute
but this peak was not suitable for analysis.
The composition of mobile phase was modified using
methanol instead of water, then the peak of terbinafine
hydrochloride appeared at a retention time of (8, 5)
minute and this peak was suitable for analysis (fig 2).
Validation of analytical method
24
Validation was based on the requirements of the USP by
studying the fallowing parameters: linearity, specificity,
precision, accuracy and recovery, limit of detection, limit
of quantification and robustness.
Figure 3: linearity of HPLC method.
Specificity
To prove that the method could determine accurately
terbinafine hydrochloride in the presence of other
components in a sample matrix a placebo sample was
injected in the HPLC apparatus there was no response
and no peak appeared at the standard retention time and
also the bias between test results obtained by analysis of
samples containing added placebo and the test results
obtained by analysis of samples without placebo was
calculated and the value did not exceed 2%.
The results of specificity are displayed in table (2).
Table 2: Specificity of the HPLC method
The average of test results with placebo
102,62%
The average of test results without placebo
102,43%
The bias
0,28%
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Int. J. Pharm. Sci. Rev. Res., 21(2), Jul – Aug 2013; nᵒ 11, 58-61
Accuracy and recovery
Accuracy was determined by applying the method to
samples of placebo to which known amounts of
terbinafine hydrochloride were added covering the
working range 50% to 150% of the standard solution,
then the recovery was calculated from the test results as
the percentage of terbinafine hydrochloride recovered by
the assay and it was 100,43% . The results of recovery are
displayed in table 3.
Table 3: Recovery of the HPLC method
Number of samples
Recovery
Relative standard deviation RSD
9
100,43%
1,63%
Precision
The precision of the method was determined by
repeatability (intraday) and intermediate precision
(intraday) studies.
Repeatability
Nine samples of terbinafine hydrochloride cream covering
the working range 50% to 150% of the standard solution
were injected in HPLC apparatus in the same day, then
the RSD of the results was calculated and it did not
exceed 2%.
ISSN 0976 – 044X
parameters. In our study flow rate was increased from 0,
4 ml / minute to 0, 6 ml / minute and this small change in
flow rate did not cause important differences on assay
value and relative retention time. The results are
displayed in table 5.
Table 5: Robustness of HPLC method.
Assay value
Relative retention
time
Table 4: Repeatability and intermediate precision of HPLC
method.
Number of samples
Average of test results
Relative standard deviation
Repeatability
Intermediate
precision
9
105,90%
1,99%
9
102,75%
2,6%
Limit of quantification
Limit of quantification which is the lowest concentration
of terbinafine hydrochloride that can be determined with
acceptable precision and accuracy by the method was (0,
2) µg /ml.
Robustness
Robustness is the capacity of the method to remain
unaffected by small and deliberate variations in method
99,66%
100,85%
1
1,001
The HPLC method developed in this study is sensitive,
specific and rapid, more over the method is simple and
uses simple regents with minimum sample preparation
procedures encouraging its application in routine analysis
of terbinafine hydrochloride in creams.
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Source of Support: Nil, Conflict of Interest: None.
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